Development of a Contemporary Animal Model of Candida albicans-Associated Denture Stomatitis Using a Novel Intraoral Denture System

Abstract

Denture stomatitis (DS) is a fungal infection characterized by inflammation of the oral mucosa in direct contact with the denture and affects up to 50% of denture wearers. Despite the prevalence, very little is known about the role of fungal or host factors that contribute to pathogenesis. Recently, we developed a novel intraoral denture system for rodent research. This denture system consists of custom-fitted fixed and removable parts to allow repeated sampling and longitudinal studies. The purpose of this study was to use this denture system to develop a clinically relevant animal model of DS. To establish DS, rats were inoculated with pelleted Candida albicans, which resulted in sustained colonization of the denture and palate for 8 weeks postinoculation. Biofilm formation on the denture was observed by week 4 and on the palate by week 6 postinoculation. Rats were monitored for clinical signs of disease by assigning a clinical score after macroscopic examination of the palate tissue according to Newton's method. By week 4 postinoculation, the majority of inoculated rats with dentures exhibited a clinical score of 1 (pinpoint erythema). By week 6 and week 8 postinoculation, increasing percentages of rats exhibited a clinical score of 2 (diffuse erythema/edema). Histological analysis of palate tissue demonstrated progressively increasing inflammatory cell recruitment throughout the time course of the infection. Palatal biofilm formation was commensurate with development of palatal erythema, which suggests a role for biofilm in the inflammatory response.

INTRODUCTION

Denture stomatitis (DS) is an inflammatory fungal infection affecting approximately 30 to 75% of otherwise healthy denture wearers and the most common form of oral candidiasis (1, 9, 10, 19, 36, 46, 50). The main causative agent of DS is Candida albicans (38). Symptoms of Candida-associated denture stomatitis range from mild to severe, including palatal edema, painful inflammation, and papillary hyperplasia (small pebble-like sores) (49). Denture stomatitis can have a negative impact on the quality of life for those affected, with very high recurrence rates despite treatment with antifungal therapy (2, 3, 5, 7, 12, 38, 47). Despite its prevalence, the pathogenesis of DS and the role of the immune response are not understood. The current dogma for the other major form of oral candidiasis, oropharyngeal candidiasis (OPC), is that a deficiency or reduction in Th1 or Th17 responses is associated with susceptibility to infection (16, 26, 37). As such, OPC predominately affects individuals with T cell deficiencies such as AIDS patients, infants, and patients receiving immunosuppressive therapy. In contrast, the majority of individuals with DS are immunocompetent. One of the few immunological studies conducted in DS patients reported Th1 cytokines in saliva; therefore, it is unlikely that the dogmatic deficiency/reduction in the Th1 response is associated with susceptibility to DS (32). In addition, elevated levels of the T cell growth factor interleukin-2 (IL-2) have been observed in patients with DS (42). Therefore, one may hypothesize that chronic T cell responses may be associated with symptomatic disease in DS.

C. albicans readily forms biofilms on denture material in vitro and in vivo, which consist of a network of hyphae and yeast encased in a polysaccharide matrix (34, 39–41). C. albicans biofilms exhibit increased resistance to drugs and antifungal host defenses (14, 23, 29, 30). Compared with free-floating C. albicans, biofilms have been shown to preferentially induce production of cytokines involved in driving Th1 or Th17 responses (14, 48). In the case of DS, it is possible that a C. albicans biofilm formed on the denture continually inoculates the host mucosa, leading to tissue-associated biofilm formation. The proinflammatory nature of the mucosal biofilm could lead to chronic inflammation that fails to clear the infection due to the presence of the contaminated indwelling device.

Previous models of DS were pioneered in the 1970s and 1980s (6, 35) with considerable research being conducted in the 1980s and 1990s (4, 8, 11–13, 33, 45). However, research since has been sparse, presumably due to the HIV epidemic and the concomitant rise in the incidence of OPC. In these animal models of DS, the investigators inserted non-custom-fitted acrylic dentures affixed to the incisors, using one rat as a template for all appliances (6, 35). There are several limitations of these DS models that have hindered progress with research aimed at understanding the host and fungal factors that influence disease progression. First, there are problems with the use of a single rat as a template for fabrication of all dentures. This could result in ill-fitting appliances and limit contact between the palate and denture. In addition, because the dentures are fixed, longitudinal analysis of fungal burden and immune responses using the same animal is not possible. Another issue is that the material used to construct the denture is currently not used clinically, and newer formulations of denture materials may result in differences between previous studies and modern studies. There was also a lack of tools available to analyze innate and adaptive immune responses. Finally, the role of fungal pathogenic traits (i.e., biofilm formation) was not considered, as mutants of C. albicans were not readily available; only one wild-type strain was used in previous studies. In addition, C. albicans fungal burden on the denture and palate was not quantified, and neither morphology nor biofilm formation was analyzed. Hence, if studies on DS are to be resurrected, given the continued wide-scale incidence of DS, it will be important to use contemporary model systems.

In order to study the immunopathogenesis of denture stomatitis, we have developed a new innovative rodent denture system that uses individual impression casting for production of custom-fitted devices made from modern denture material (31). This denture system was engineered with both fixed and removable portions to facilitate longitudinal studies (patent pending). In these studies, we describe the optimization of a model of Candida-associated DS using this novel denture system, which results in palatal inflammation and clinical signs of disease (palate erythema and edema) in immunocompetent rats following both denture and palate biofilm formation.

MATERIALS AND METHODS

Animals.

Male retired breeder Wistar rats (400 to 800 g) were purchased from Charles River Laboratories. All rats were maintained at an American Association for the Accreditation of Laboratory Animal Care (AAALAC)-accredited animal facility at Louisiana State University Health Sciences Center (LSUHSC) and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals under a protocol proposal approved by the LSUHSC Institutional Animal Care and Use Committee. The animals were fed gel diet 76A (clear H₂O).

Human saliva collection.

Saliva was obtained from 10 healthy volunteers who were enrolled in accordance with the stated guidelines of the Institutional Review Board of Louisiana State University Health Sciences Center. Participants included 8 females and 2 males, with ages ranging from 20 to 45 years. Exclusion criteria included any form of oral pathology, oral infections, periodontitis, or gingivitis. Once informed consent was obtained from each subject, saliva was collected from each individual at approximately the same time of day (late afternoon). For this, subjects were asked to expectorate 10 ml of unstimulated whole saliva into a 50-ml sterile plastic centrifuge tube. Following collection, whole saliva was clarified by centrifugation for 5 min at 800 × g at 4°C. The supernatants were collected and sterile filtered using a 45-μm low-protein binding filter. The filtered saliva was then aliquoted into 1-ml volumes and frozen at −80°C until use.

Candida albicans strains.

Strain DAY185 is a complemented prototroph derived from a triple auxotrophic strain (BWP17; parent = SC5314), which is frequently used to produce knockout and complemented strains of C. albicans. For each experiment, strains were subcultured from freezer stocks onto Sabouraud dextrose agar (SDA) plates and incubated at 30°C overnight. Cultures were washed in 1× phosphate-buffered saline (PBS), counted, and diluted in sterile 1× PBS for inoculation. C. albicans was cultured overnight at 30°C, washed in 1× PBS, centrifuged, weighed, and suspended in PBS at various concentrations or as a paste at various milligram quantities. To determine fungal concentration, the inocula were analyzed by quantitative culture assay.

Quantitative culture assay.

Samples were vortexed, and 10-fold dilutions were made in sterile 1× PBS and plated onto SDA plates. Plates were incubated at 37°C overnight, and resulting colonies were counted visually and expressed as CFU.

In vitro model of denture biofilm formation.

Polymethyl methacrylate (PMMA) acrylic resin (self-curing denture; Lang Dental, Wheeling, IL) was prepared according to the manufacturers' instructions and spread onto glass microscope slides. The PMMA was scored to make uniformly sized squares and allowed to cure. Denture chips were placed into sterile 6- or 12-well plates. For inoculation, C. albicans strain DAY185 was diluted in sterile 1× PBS or filtered whole saliva to a final concentration of 1 × 10⁴ CFU/ml. For inoculation, 100 μl of C. albicans suspension was added to the surface of the denture chip. The sample was incubated at 37°C for 3 h and then rinsed gently with 1× PBS and submerged in saliva for an additional 24 h at 37°C before being processed for microscopic analysis.

Ex vivo model of mucosal biofilm formation.

Rats were euthanized, and palatal tissue was excised using a sterile scalpel. Palate tissue was placed into sterile 12-well plates containing sterile 1× PBS. Tissues were not submerged, and the epithelial surface remained air exposed. For inoculation, C. albicans strain DAY185 was diluted in sterile 1× PBS or filtered whole saliva to a final concentration of 1 × 10⁴ CFU/ml. For inoculation, 100 μl of C. albicans suspension was added to the surface of the palate tissue. The sample was incubated at 37°C for 3 h and then rinsed gently with 1× PBS and submerged in saliva for an additional 24 h at 37°C before being processed for microscopic analysis.

In vivo rat denture stomatitis model.

Our laboratory recently designed a new custom-fitted denture system in rats (patent pending) (31). This innovative rodent denture system was engineered with both fixed and removable portions. For custom fitting, impressions were taken from individual rats, using light-body vinyl polysiloxane (VPS) impression material (Aquasil Ultra LC; Dentsply Caulk) applied to the maxillary palate. A wooden applicator was inserted into the VPS to facilitate removal of the solidified impression (Fig. 1A and B). Impressions were used to produce stone mold templates for production of the fixed and removable dentures (Fig. 1C and D). For installation, the fixed portion, which is embedded with magnets, is anchored to the rear molars of the rat by orthodontic wires (Fig. 1E). The removable portion, which fits over the anterior palate, attaches magnetically to the fixed portion via an embedded metal rod (Fig. 1F). The removable portion can easily be detached for sampling and replaced, which allows for longitudinal analyses.

Fig 1.

Removable intraoral rodent denture system for rodent research. Individual impressions are made for each rat using vinyl polysiloxane impression material (A and B). Molds are made from the impressions (C and D), which are used to construct the fixed (C) and removable (D) portions of the denture system. The fixed portion is anchored to the rear molars and contains embedded magnets (E). The removable portion fits over the proximal rat palate, which is connected to the fixed portion via a magnet (F).

For inoculation, rats were first anesthetized by intraperitoneal injection with 90 mg ketamine/kg of body weight plus 10 mg/kg xylazine. Two methods of inoculation were tested: inoculation with liquid suspension (100 μl of a C. albicans suspension was pipetted under the removable portion of the denture system) or inoculation with pelleted yeast paste (the removable portion of the denture system was detached, the paste was spread on the palate of the rat, and the denture was replaced). Paste quantities tested included 25 mg (≈5 × 10^9 CFU) and 50 mg (≈1 × 10^10 CFU). For quantification of fungal burden on the denture and palate tissue, animals were anesthetized. The removable portion of the denture was detached, and the intaglio surface of the denture and the palate were each swabbed with individual sterile cotton-tipped applicators. Every 2 weeks, the buccal mucosa, tongue, and gingiva were also sampled. The applicators were vortexed in 1 ml of 1× PBS, and fungal burden was analyzed by quantitative culture assay. Palatal tissue underlying the denture system was evaluated visually and scored for severity of inflammation, according to Newton's method (34a): 0, no inflammation; 1, pinpoint erythema; 2, diffuse erythema and edema; 3, diffuse erythema/edema and papillary hyperplasia. For microscopy and histology, palates were excised and dentures were removed from euthanized animals at 2-week intervals for 8 weeks. Controls consisted of naïve animals, uninoculated animals with the denture system, and inoculated animals without the denture system.

Confocal microscopy (CM).

Excised palate tissue or denture material was placed in 12-well dishes and stained for 20 min at room temperature with 1 mg/ml calcofluor white (Fluka), which stains the fungal cell wall, and 50 μg/ml concanavalin A-Texas Red conjugate (ConA-TR; Molecular Probes), which is a lectin that stains the biofilm extracellular matrix (ECM). Tissue or denture material was removed from staining wells, gently rinsed with 1× PBS to remove exogenous stain, and placed onto glass microscope slides. Samples were analyzed using an Olympus Fluoview FV100 confocal microscope and Fluoview software.

SEM.

Excised palate tissue or dentures were fixed in 2.5% glutaraldehyde overnight at 4°C. Samples were rinsed twice in 1× PBS before postfixing in 1% osmium tetroxide for 5 to 10 min at room temperature. Following two washes in 1× PBS, the samples were dehydrated in 20% ethanol for 1 min, followed by three successive 1-min incubations in 40%, 60%, 80%, 95%, and 100% ethanol allowed to dry overnight. All samples were fixed to scanning electron microscopy (SEM) studs with double-sided magnetic tape and carbon coated. Specimens were viewed at 100× to 1,000× magnifications on a Hitachi S-2700 scanning electron microscope (LSUHSC Imaging Core) with the voltage set to 15 kV.

Histology.

Excised palate tissue was embedded in optimal cutting temperature (OCT) medium and flash frozen over dry ice. The samples were stored at −80°C. The tissue was cut into 5- to 6-μm sections using a cryostat and placed on glass microscope slides, fixed in acetone for 10 min, and stored at −20°C. Fixed slides were stained with hematoxylin and eosin.

RESULTS

C. albicans biofilm formation on denture material and palate tissue ex vivo.

The ability of C. albicans strain DAY185 to form biofilms in saliva on the PMMA denture material and on excised rat palatal tissue was first tested to demonstrate the feasibility of the in vivo model. Biofilm formation was analyzed 24 and 48 h postinoculation via SEM and CM. Ample biofilm growth was observed by 24 h on both denture material and excised palatal tissue (Fig. 2A to D). The C. albicans biofilms exhibited typical architecture, consisting of both yeast and hyphal forms surrounded by an extracellular matrix (ECM) as visualized with confocal microscopy by ConA-TR staining, which stained both cell wall-associated and diffuse intercellular ECM (Fig. 2C and D). No colonization or biofilm formation was seen on uninoculated denture material or excised palatal tissue (data not shown).

Fig 2.

In vitro biofilm growth. A 24-hour biofilm was grown on denture material or rat palate tissue. C. albicans DAY185 (1 × 10⁶ CFU) in saliva was used to inoculate denture material (A and C) or excised rat palate tissue (B and D) and incubated at 37°C for 24 h to allow biofilm growth. The sample was then processed for either SEM (A and B) or confocal microscopy (C and D). The figure shows a representative image of 4 repeats. Magnification, ×500.

Establishment of C. albicans colonization in a novel rat denture stomatitis model.

To establish an in vivo DS model, immunocompetent rats were fitted with the fixed and removable denture system and allowed to acclimate for 2 weeks. We tested several methods of inoculation to determine conditions that produce consistent fungal colonization of the denture and palate. In initial experiments, rats were inoculated with a liquid suspension of C. albicans DAY185 containing 10⁶, 10⁷, or 10⁸ CFU in 100 μl of PBS. This method resulted in low and inconsistent colonization over a 2-week period (data not shown). Alternatively, inoculation was performed with C. albicans as a paste in various quantities (10, 25, and 50 mg) and applied to the palate while the removable portion of the denture was removed. Following the immediate reinstallation of the removable denture piece, colonization was monitored weekly over a 4-week period. The colonization of the removable rat denture and the palate was detectable and consistent over time with the highest levels of colonization using a 25- or 50-mg paste inoculum (data not shown).

Using a 50-mg paste inoculum, a complete analysis was performed over a 4-week period including inoculated control animals and inoculated animals not fitted with a denture. We observed stable levels of colonization of the palate and denture over the 4-week period in animals with a denture (Fig. 3A and B). Uninoculated control animals showed no evidence of colonization regardless of the presence or absence of a denture. Inoculated control animals without a denture had low levels of colonization over the 4-week period (Fig. 3A and B). Trace colonies of Candida were detected at other sites in the oral cavity (i.e., tongue and buccal mucosa) periodically early postinoculation (1 to 2 weeks) but not at later time periods (data not shown).

Fig 3.

Candida colonization in vivo requires the presence of the denture. Rats were inoculated with 25 mg of Candida albicans DAY185. Swabs of both the palate (A) and the denture (B) were taken weekly and processed for quantitation of fungal burden over a period of 4 weeks. Controls included uninoculated animals with and without the denture system and inoculated animals without the denture system. n = 4 to 6 rats.

C. albicans forms biofilm on denture and palate in vivo.

Having established optimal inoculation conditions for sustained colonization, we initiated long-term experiments in immunocompetent rats to monitor biofilm formation and onset of clinical signs of DS. Dentures and harvested palates were collected from 50-mg paste-inoculated animals and analyzed at 2, 4, 6, and 8 weeks postinoculation for colonization and biofilm formation. Biofilm formation was evaluated using SEM and CM. Colonization was consistent at a high level over the 8-week period in inoculated animals with the denture (Fig. 4). Colonization was not observed in uninoculated animals with a denture at 8 weeks postinoculation or in inoculated animals without a denture at 8 weeks (data not shown). The lack of colonization also precluded any biofilm formation. Biofilm formation on the palate was not evident at 2 or 4 weeks postinoculation, with only yeast forms visible and no discernible ECM (Fig. 5 and 6). However, by week 6 postinoculation, hyphae were present and the surface of the biofilm was coated with ECM as evidenced by ConA staining observed in CM micrographs and the presence of a film covering cellular structures observed in SEM micrographs. The presence of mature biofilms on the palate persisted at 8 weeks postinoculation (Fig. 5 and 6). Biofilm formation on the denture was evident at 4 weeks postinoculation and continued to mature through 8 weeks postinoculation as shown by both SEM and CM (Fig. 5 and 6). The appearance of the in vivo palate and denture biofilms was nearly identical to that in the in vitro denture and ex vivo palate explant models, confirming the biofilm phenotype in the in vivo model (Fig. 2, 5, and 6). Biofilm growth was unaffected by the biweekly swabs of the denture surface or the palate, as similar levels of colonization were detected at 8 weeks postinoculation whether or not the removable denture was swabbed biweekly or not (data not shown).

Fig 4.

Candida colonization on the denture and palate is sustained for 8 weeks postinoculation. Rats were inoculated with 25 mg of Candida albicans DAY185. The palate and the denture were swabbed biweekly to analyze fungal burden. Results represent the averages of 3 independent experiments. n = 6 to 10 rats.

Fig 5.

Scanning electron microscopy analysis of C. albicans biofilm formation on the denture and palate in vivo. Palate tissue and denture were harvested from inoculated rats and were processed for SEM. Images are representative of 3 independent repeats. Bar, 50 μm.

Fig 6.

Confocal microscopy analysis of C. albicans biofilm formation on the denture and palate in vivo. Palate tissue and denture were harvested from inoculated rats and were stained with calcofluor white (cell wall; blue) and ConA-TR (ECM; red). Images are representative of 3 independent repeats. Magnification, ×600.

Palate tissue was also processed for histology at 2-week intervals (Fig. 7). Hematoxylin and eosin staining of the palate showed evidence of inflammation over the course of the infection. Slight but noticeable changes in the histology of the palates were observed by 4 weeks postinoculation. The most severe inflammation was evident at 6 and 8 weeks postinoculation. No marked differences were observed between the tissue inflammation at week 6 and week 8 postinoculation. Control animals that were inoculated without a denture or uninoculated with a denture showed no evidence of inflammation over the 8-week period (Fig. 7). We also did not observe any evidence of the fungal biofilm by histology, which is likely due to removal of the superficial biofilm during processing. The histology results correlated with the clinical scores of disease. In uninoculated control rats, the palate tissue appears white with defined palatal ridges indicating an absence of edema, which is defined as a clinical score of 0 (Fig. 8B). In infected rats, clinical signs of DS became evident at 4 weeks postinoculation, with the majority of animals having a clinical score of 1 (pinpoint erythema and edema) (Fig. 8A and C). By weeks 6 and 8 postinoculation, increasing numbers of rats progressed to a clinical score of 2 (diffuse erythema and edema) (Fig. 8A and D). No animals had a clinical score of 3 by week 8, and uninoculated animals with a denture never showed clinical signs of disease (Fig. 8A and data not shown).

Fig 7.

Histological analysis of palatal inflammation. Palate tissue was harvested from uninoculated control or inoculated animals with or without a denture and processed for histology. Slides were stained with hematoxylin and eosin. Images are representative of 3 independent repeats. Magnification, ×200.

Fig 8.

Development of palatal inflammation, erythema, and edema. (A) Clinical score: 0, no signs of disease; 1, pinpoint erythema/edema; 2, diffuse erythema/edema. Results represent the averages of 3 independent experiments. (B) Uninoculated control rat palate. (C) Inoculated rat palate tissue underlying denture at week 4 postinoculation. The arrow points to an area of pinpoint erythema and edema (clinical score, 1). (D) Inoculated rat palate tissue underlying denture at week 6 postinoculation. Arrows point to areas of diffuse erythema and edema; note that the palatal ridges are not visible due to swelling (clinical score, 2). Images are representative of 3 independent repeats.

DISCUSSION

Our goal for these studies was to develop a new contemporary rat model of DS in immunocompetent animals. The new model utilizes a fixed and removable denture system (patent pending) (31) that allows for easy sampling and longitudinal studies, both of which were missing in previous models of DS (6, 35, 44). Moreover, the denture system is fabricated from materials presently used in clinical dentistry and is custom fitted to each anima to facilitate optimal contact between the denture and palate.

Attempts to induce denture/palatal colonization and clinical signs of disease using liquid inocula of C. albicans proved to be ineffective and inconsistent in this model. Although low levels of colonization were observed for 1 to 2 weeks postinoculation, there was no evidence of either biofilm formation or clinical disease on the denture or palate. In early rat models of DS, animals were inoculated with 30 mg of pelleted C. albicans blastospores, which was spread onto the denture (35). Therefore, we tested a range of weights of pelleted C. albicans in this model and found that a 25- or 50-mg pellet (≈5 × 10^9 or ≈1 × 10^10 CFU, respectively) was optimal for establishing consistently high colonization. It is important to note that this high inoculum did not result in formation of white lesions typical of OPC on the palate or tongue or lead to extraoral dissemination, which would compromise the clinical relevance of this model. The stringency of the model was revealed through the use of experimental controls (i.e., noninoculation with and without a denture and inoculation without a denture) that showed little or no colonization and only for a short period of time (inoculation without a denture).

After confirmation that C. albicans could form biofilms in saliva on both denture material and rat palate explants in vitro, the first evidence of in vivo biofilm occurred on the denture at 4 weeks postinoculation. By 6 weeks postinoculation, biofilms were evident on both the denture and the palate by SEM and CM. The majority of the animals exhibited a clinical score of 1 by week 4, coinciding with the formation of the biofilm on the denture. More severe clinical signs (score of 2) were observed at 6 weeks postinoculation when biofilm was present on both the denture and the palate. Histology of the palate tissue revealed the presence of increasing numbers of inflammatory cells underlying the mucosal epithelium during weeks 4 to 8. It is interesting that punctate erythema was observed (clinical score of 1), prior to the presence of biofilm on the palate. This suggests that biofilm formation on the tissue is not required to initiate clinical disease but can accelerate it once present. Alternatively, a clinical score of 1 may be more or less due to irritation caused by the presence of the yeast (biofilm on the denture and palatal colonization), whereas advanced clinical scores (2 or 3) require biofilm formation on the palate. It should be noted that uninoculated animals with a denture did not develop erythema or edema, ruling out any effect of the denture alone. Overall, the presence of both denture and palatal biofilms along with palatal inflammation in immunocompetent animals is consistent with clinical DS in humans. Therefore, this model can be used to study the role of both microbial and host factors in pathogenesis of DS.

Recently, a rat model of acute C. albicans denture-associated biofilm infection was reported (34). In this model, rats were immunosuppressed with cortisone, which is often used in animal models of oropharyngeal candidiasis (OPC) to facilitate tissue infection and invasion. Denture devices were engineered with a 1-mm gap between the palate and denture for inoculation, which limits contact between the denture and palate. Animals were inoculated with a liquid suspension of C. albicans and followed for 72 h postinoculation. While C. albicans biofilms formed on denture material in vivo after 48 h, there was no evaluation of palatal biofilm formation or clinical score and histology showed no evidence of palatal inflammation. However, there was significant hyphal invasion of both palate and tongue tissues, characteristic of OPC models. Therefore, the model does not simulate a clinically relevant form of DS. Moreover, DS is not associated with immune deficiency or invasive infection (28, 46). On the other hand, this model is potentially quite useful for quickly evaluating the ability of different C. albicans strains to form biofilms on denture material in an in vivo setting, with the caveat that the ability of the immune system to interfere with biofilm formation could not be evaluated. In comparison, the model described here simulates the clinical condition of DS, in terms of both a time perspective and clinical scores, etc., and serves best to study the pathogenesis and role of biofilm formation in disease.

The significance of the biofilm in this clinical disease remains in question. Aside from the potential role of the palate biofilm in accelerating disease, there is also the question of whether the denture biofilm promotes palate biofilm formation through continual seeding of the tissue. However, to test/confirm/prove this hypothesis, studies using biofilm-deficient mutants will need to be performed. Previous experiments in the acute denture biofilm model developed by Nett et al. demonstrated that a biofilm-deficient mutant (Δbcr1/Δbcr1) exhibited reduced adherence but was still able to colonize the denture at relatively high levels (34). Therefore, it is feasible to test this mutant in this model of Candida-associated DS. Other groups have reported bacteria associated with a C. albicans oral mucosal or device-associated biofilm (21, 34). We also observed bacteria associated with the C. albicans biofilm by SEM in this model (data not shown). It is unclear whether polymicrobial biofilms contribute to or alter the pathogenesis of DS. However, using an in vitro oral mucosa model, it was shown that polymicrobial biofilms of C. albicans and oral streptococci result in increased fungal invasion (20). Therefore, this warrants further examination in DS. In addition, up to 50% of DS patient samples contain more than one species of Candida, very often a combination of C. albicans and Candida glabrata (22, 47). Clinical evidence points to a role for mixed-species biofilms of C. albicans and C. glabrata in increasing severity of disease (15). C. glabrata is also inherently more resistant to antimicrobial drugs, including DS patient isolates (18, 22). Relating the clinical evidence to this DS model will be an important focus of future experiments.

Likewise, little is known about the pathogenesis of DS. Most patients with DS are otherwise immunocompetent. Therefore, the pathogenesis may involve development of a chronic, and possibly aggressive, inflammatory response that occurs due to the continuous seeding of Candida from the denture to the palate. Currently, it is unclear whether innate and/or adaptive immunity fuels the response and whether immune deficiency could exacerbate or alleviate disease. A report by Gasparoto and coworkers suggested that a deficiency in neutrophil function may play a role in DS because a lower number of neutrophils were found in the saliva of DS patients (25). This may also have been related to the reduced phagocytic activity of systemic neutrophils from elderly patients for Candida regardless of disease (24). Alternatively, there may be a role for T cells in facilitating chronic inflammation. It is known that Th17 responses are induced in response to oral C. albicans infections and required for protection against OPC (16, 17, 27, 32). However, Th17 responses have also been implicated in exacerbation of candidal infections (i.e., gastrointestinal [GI] tract) (43). Hence, either deficient or chronic Th17 responses could be playing a role in DS. Future studies in this DS model will include detailed and kinetic analysis of the immune mediators and inflammatory cell types observed in the histology as well as experiments targeting components of the immune response (innate and T cell mediated) to determine their role in pathogenesis.